1. Field of the Invention
The present invention relates to novel parathyroid hormone peptide (PTH) derivatives. In particular, the invention relates to PTH derivatives having one or more amino acid substitutions that confer PTH-1 receptor agonist or antagonist properties to the derivatives.
2. Description of Related Art
Full understanding of the complex biological roles of parathyroid hormone (PTH) and efforts to utilize its therapeutic potential require dissection of the multiple signaling patterns and cellular pathways of action of the hormone. PTH binds and activates specific receptors in renal and osseous target cells that also recognize PTH-related peptide (PTHrP) (Kronenberg, H., et al., “The PTH/PTHrP receptor: one receptor for two ligands,” in Genetics of Endocrine and Metabolic Disorders, Thakker, R., ed., Chapman & Hall, London (1997), pp. 389-420). In renal and osteoblastic cell lines, PTH triggers several parallel intracellular signaling responses, including activation of adenylyl cyclase (AC), protein kinase A (PKA), phospholipase C (PLC) and protein kinase C (PKC) and generation of second messengers such as cyclic AMP (cAMP), inositol trisphosphate (IP3), diacylglycerol and increased cytosolic free calcium (Cai++) (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. USA. 89(7):2732-2736 (1992); Azarani, A., et al., J. Biol. Chem. 271(25): 14931-14936 (1996); Bringhurst, F. R., et al., Endocrinology 132(5):2090-2098 (1993); Civitelli, R., et al., Am. J. Physiol. 255(5 Pt 1):E660-667 (1988); Donahue, H. J., et al., J. Biol. Chem. 263:13522-13527 (1988); Dunlay, R., and Hruska, K., Am. J. Physiol. 258(2 Pt 2):F223-231 (1990); Fujimori, A., et al., Endocrinology 128(6):3032-3039 (1991); Fujimori, A., et al., Endocrinology 130(1):29-36 (1992); Guo, J., et al., Endocrinology 136(9):3884-3891 (1995); Janulis, M., et al, Endocrinology 133:713-719 (1993); Jouishomme, H., et al., J. Bone Miner. Res. 9(6):943-949 (1994); Juppner, H., et al., Science 254(5034):1024-1026 (1991); Pines, M., et al., Bone 18(4):381-389 (1996); Seuwen, K., et al., Brit. J. Pharm. 114(8):1613-1620 (1995); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995).
To date, two structurally related but distinct species of PTH receptors have been cloned (Abou-Sarnra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Usdin, T. B., et al., J. Biol. Chem. 270(26):15455-15458 (1995); Schipani, E., et al., Endocrinology-132(5):2157-2165 (1993)). The first of these, type A, was isolated from both bone and kidney cells and shown to transduce multiple signaling responses to PTH-(1-34) or PTHrP(1-36) when heterologously expressed in cells that lack endogenous type 1 PTH/PTHrP receptors (hereinafter PTH-1 receptors) (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Azarani, A., et al., J. Biol. Chem. 271(25):14931-14936 (1996); Bringhurst, F. R., et al., Endocrinology 132(5):2090-2098 (1993); Guo, J., et al., Endocrinology 136(9):3884-3891 (1995); Pines, M., et al., Bone 18(4):381-389 (1996); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997); Schneider, H., et al., Eur. J. Pharm. 246(2):149-155 (1993)).
Previous efforts to define the contributions of specific regions of the PTH molecule to its binding and signaling properties have been undertaken mainly by use of complex in vivo bioassays, organ cultures, isolated cell membranes or cell lines, generally of rodent origin, that may express more than one type of endogenous PTH-1 receptors (Janulis, M., et al., Endocrinology 133:713-719 (1993); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995); Yamamoto, S., et al., Endocrinology 138:2066-2072 (1997); Jouishomme, H., et al., Endocrinology 130(1):53-60 (1992); Segre, G. V., et al., J. Biol. Chem. 254:6980-6986 (1979); Tregear, G. W., and Potts, J. T., Jr. Endocr. Res. Commun. 2:561-567 (1975); Takasu, H., et al., Endocrinology 137(12):5537-5543 (1996); Orloff, J. J., et al., Am. J. Physiol. 262(5 Pt 1):E599-607 (1992)).
Early structure/function studies of bovine PTH-(1-34), performed with isolated renal membranes, identified the key role of the carboxyl(C)-terminal bPTH-(25-34) region for receptor binding and of the amino(N)-terminus (i.e., Ser1) for AC activation (Segre, G. V., et al., J. Biol. Chem. 254:6980-6986 (1979); Tregear, G. W., and Potts, J. T., Jr. Endocr. Res. Commun. 2:561-567 (1975)). Later work conducted in vitro with intact renal tubules or with cultured renal or bone cells, however, indicated that N-truncated analogs such as PTH-(3-34), although unable to stimulate AC, could fully activate PKC and could regulate certain PKC-dependent distal biologic responses (Janulis, M., et al., Endocrinology 133:713-719 (1993); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995); Jouishomme, H., et al., Endocrinology 130(1):53-60 (1992)). Amino-truncated analogs of PTH-(1-34) also were found to increase PLC activity or Cai++ in some cells (Donahue, H. J., et al., J. Biol. Chem. 263:13522-13527 (1988); Fujimori, A., et al., Endocrinology 128(6):3032-3039 (1991); Siegfried, G., et al., Endocrinology 136(3):1267-1275 (1995)) though not in others (Reid, I. R., et al., Am. J. Physiol. 253(1 Pt 1):E45-51 (1987); Tamura, T., et al., Biochem. Biophys.,Res. Commun. 159:1352-1358 (1989)). Studies of the signaling properties of the cloned PTH-1 receptor have focused almost exclusively upon activation of AC, PLC or Cai++ (Abou-Samra, A. B., et al., Proc. Natl. Acad. Sci. U.S.A. 89(7):2732-2736 (1992); Bringhurst, F. R., et al., Endocrinology 132(5):2090-2098 (1993); Guo, J., et al., Endocrinology 136(9):3884-3891 (1995); Pines, M., et al., Bone 18(4):381-389 (1996); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997); Schneider, H., et al., Eur. J. Pharm. 246(2):149-155 (1993)), although stimulation of PKC and of PKC-dependent ion transport by hPTH(1-34), hPTH-(3-34) and other hPTH fragments was reported in CHO cells transfected with rat PTH-1 receptor cDNA (Azarani, A., et al., J. Biol. Chem. 271(25):14931-14936 (1996)).
Collectively, these observations have engendered the concept that the structural determinants for activation of AC/PKA signaling are distinct from those required for activation of PLC or PKC and that these reside, respectively, within the N- and C-terminal domains of PTH-(1-34) (Jouishomme, H., et al, J. Bone Miner. Res. 9(6):943-949 (1994); Tregear, G. W., and Potts, J. T., Jr. Endocr. Res. Commun. 2:561-567 (1975); Whitfield, J. F., and Morley, P. Trends Pharm. Sci. 16(11):382-386 (1995)). In particular, the region hPTH-(29-32) was identified specifically as a critical PKC activation domain (Jouishomme, H., et al., J. Bone Miner. Res. 9(6):943-949 (1994); Whitfield, J. F., and Morley, P. Trends Pharm. Sci. 16(11):382-386 (1995)).
Compared with what is known from these studies of the rat PTH-1 receptor, much less information is available regarding the structural features of human PTH required for binding to the human PTH-1 receptor or for activation of its various signaling modes. Alanine-scanning mutagenesis has highlighted the importance of the C-terminal portion of hPTH-(1-34) for binding to the rat PTH-1 receptor (30). Functional studies of transfected human PTH receptors in COS-7 or HEK 293 cells have confirmed that hPTH-(1-34) activates AC and Cai++, although stimulation of PLC was not observed consistently and responses that were reported were modest (Pines, M., et al., Bone 18(4):381-389 (1996); Seuwen, K., et al., Brit. J. Pharm. 114(8):1613-1620(1995); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997); Schneider, H., et al., FEBS Lett. 351(2):281-285 (1994); ). The effects of hPTH-(3-34) on Cai++ are similarly controversial (Pines, M., et al., Bone 18(4):381-389 (1996); Jobert, A.-S., et al., Endocrinology 138(12):5282-5292 (1997)), while the roles of other regions of the hPTH-(1-34) molecule in signaling via the human PTH-1 receptor have not been systematically addressed. Synthetic hPTH-(1-30)NH2, hPTH-(1-29)NH2, hPTH-(1-28)NH2, hPTH-(1-27)NH2, and hPTH-(1-26)NH2 were each incapable of stimulating the activity of membrane-bound PKCs in osteoblast-like ROS 17/2 cells (Neugebauer et al. (Biochem 34: 8835-8842 (1995)).